The invention relates to a method of affecting or modulating a pathway in a cell which is regulated by the binding of a transmembrane ligand for an Elk-related receptor tyrosine kinase and an oligomerized Elk-related receptor tyrosine kinase; a method of identifying substances which affect the binding of a transmembrane ligand for an Elk-related receptor tyrosine kinase and an oligomerized Elk-related receptor tyrosine kinase; and to methods and pharmaceutical compositions using oligomerized Elk-related receptor tyrosine kinases, and substances identified using a method of the invention.
Receptor tyrosine kinases play essential roles in cellular signalling events. The largest known family of receptor tyrosine kinases is the Eph subfamily of receptor tyrosine kinases. Eph subfamily tyrosine kinases have been implicated in the control of axon guidance and fasciculation1-7, in regulating cell migration8, and in defining compartments in the developing embryo9-11. Efficient activation of Eph receptors generally requires that their ligands be anchored to the cell surface, either through a transmembrane (TM) region or a glycosyl phosphatidylinositol (GPI) group12. These observations have suggested that Eph receptors can transduce signals initiated by direct cell-cell interactions. Genetic analysis of Nuk, a mouse Eph receptor that binds TM-ligands, has suggested that these ligands have a signalling function6.
Challenging cells expressing the transmembrane (TM)-ligands, Elk-L or Htk-L, with the clustered extracellular domain of Nuk was found to induce phosphorylation of the ligands on tyrosine, a process which is mimicked both in vitro and in vivo by an activated Src tyrosine kinase. Co-culturing of cells expressing a TM-ligand with cells expressing Nuk also was shown to lead to tyrosine phosphorylation of both the ligand and Nuk. Therefore TM-ligands are associated with a tyrosine kinase, and are inducibly phosphorylated upon binding the Nuk receptor, in a fashion reminiscent of cytokine receptors. Furthermore, it was shown that TM-ligands, as well as Nuk, are phosphorylated on tyrosine in mouse embryos, indicating that this is a physiological process. These experimental results confirm that Eph receptors and their TM-ligands mediate bi-directional cell signalling.
Therefore, the present invention provides a method of modulating the biological activity of a transmembrane ligand for an Elk-related receptor tyrosine kinase in a cell expressing the transmembrane ligand comprising forming a complex between a purified and isolated oligomerized Elk-related receptor tyrosine kinase, or an isoform or an extracellular domain of the Elk-related receptor tyrosine kinase, and the transmembrane ligand expressed on the cell, thereby modulating the biological activity of the transmembrane ligand.
The present invention also provides a method of affecting or modulating a pathway regulated by a transmembrane ligand for an Elk-related receptor tyrosine kinase in a cell expressing the transmembrane ligand, comprising forming a complex between a purified and isolated oligomerized Elk-related receptor tyrosine kinase, or an isoform or an extracellular domain of the Elk-related receptor tyrosine kinase, and a transmembrane ligand expressed on the cell, thereby affecting or modulating a pathway in the cell which is regulated by the transmembrane ligand.
The invention also provides a method for evaluating a substance for its ability to modulate the biological activity of a transmembrane ligand for an Elk-related receptor tyrosine kinase in a cell expressing the transmembrane ligand comprising the steps of:
(a) contacting an oligomerized Elk-related receptor tyrosine kinase, or an isoform or an extracellular domain of the Elk-related receptor tyrosine kinase; a transmembrane ligand which binds to the Elk-related receptor tyrosine kinase to form a receptor-ligand complex, wherein the transmembrane ligand is a transmembrane ligand for an Elk-related receptor tyrosine kinase expressed on a cell; and, a test substance, under conditions which permit the formation of receptor-ligand complexes;
(b) assaying for receptor-ligand complexes, free Elk-related receptor tyrosine kinase, or non-complexed transmembrane ligand, or for activation of the transmembrane ligand; and
(c) comparing to a control to determine if the substance inhibits or enhances the binding of the Elk-related receptor tyrosine kinase and transmembrane ligand, and thereby modulates the biological activity of the transmembrane ligand.
The invention also provides a method for identifying a substance which affects or modulates a pathway regulated by a transmembrane ligand for an Elk-related receptor tyrosine kinase in a cell expressing the transmembrane ligand, comprising the steps of:
(a) contacting an oligomerized Elk-related receptor tyrosine kinase, or an isoform or an extracellular domain of the Elk-related receptor tyrosine kinase; a transmembrane ligand which binds to the Elk-related receptor tyrosine kinase to form receptor-ligand complexes which activate a pathway regulated by a transmembrane ligand for an Elk-related receptor tyrosine kinase in a cell expressing the transmembrane ligand; and a test substance under conditions which permit the formation of receptor-ligand complexes;
(b) assaying for receptor-ligand complexes, free Elk-related receptor tyrosine kinase, or non-complexed transmembrane ligand, or for activation of the transmembrane ligand; and
(c) comparing to a control to determine if the substance inhibits or enhances the binding of the Elk-related receptor tyrosine kinase and transmembrane ligand, and thereby affects or modulates the pathway.
The ability of a substance to inhibit or enhance the binding of an oligomerized Elk-related receptor tyrosine kinase and transmembrane ligand correlates with the ability of the substance to inhibit or enhance the biological activity of the transmembrane ligand, including the signal transduction activities of the ligand, and in particular the activation of a pathway regulated by the ligand.
The invention also contemplates a method for evaluating a substance for its ability to inhibit or enhance the interaction of an oligomerized Elk-related receptor tyrosine kinase, or an isoform, or an extracellular domain of the Elk-related receptor tyrosine kinase, and a transmembrane ligand for an Elk-related receptor tyrosine kinase expressed on a cell comprising the steps of:
(a) providing a reporter gene operably linked to a DNA binding site for a transcriptional activator;
(b) providing a first hybrid protein comprising the transmembrane ligand in polypeptide linkage to a DNA binding domain of the transcriptional activator;
(c) providing a second hybrid protein comprising an oligomerized Elk-related receptor tyrosine kinase or an isoform or an extracellular domain of the Elk-related receptor tyrosine kinase in polypeptide linkage to an activation domain of the transcriptional activator; under conditions where the transmembrane ligand and oligomerized Elk-related receptor tyrosine kinase can bind and thereby reconstitute the transcriptional activator which induces transcription of the reporter gene;
(d) administering a test substance; and
(e) monitoring expression of the reporter gene, wherein a decrease in expression is an indication that the substance inhibits the interaction of the transmembrane ligand and oligomerized Elk-related receptor tyrosine kinase, and an increase in expression is an indication that the substance enhances the interaction of the transmembrane ligand and oligomerized Elk-related receptor tyrosine kinase. In an alternate method, the oligomerized Elk-related receptor tyrosine kinase is linked to the DNA binding domain, and the transmembrane ligand is linked to the activation domain.
In another aspect, the invention features an antibody preparation which specifically binds to a receptor-ligand complex comprising an oligomerized Elk-related receptor tyrosine kinase, or an isoform or an extracellular domain of the kinase, and a transmembrane ligand for an Elk-related receptor tyrosine kinase.
In another aspect, the invention features a method of purifying a compound which inhibits or enhances the binding of an oligomerized Elk-related receptor tyrosine kinase, or an isoform or an extracellular domain of the kinase, and a transmembrane ligand for an Elk-related receptor tyrosine kinase comprising contacting the compound with one of the ligand or receptor; and, isolating the compound by its binding affinity for the ligand or receptor.
The substances, and compounds obtained using the methods of the invention and the antibodies specific for receptor-ligand complexes may be used to modulate the biological activity of a transmembrane ligand for an Elk-related receptor tyrosine kinase in a cell expressing the transmembrane ligand, including inhibiting or enhancing signal transduction activities of the transmembrane ligand, and in particular modulating a pathway regulated by the transmembrane ligand.
The invention still further provides a method for affecting or modulating neuronal development or regeneration in a subject comprising administering to a subject an effective amount of a purified and isolated oligomerized Elk-related receptor tyrosine kinase, or an isoform or an extracellular domain thereof, a substance or compound identified using a method of the invention, or an antibody specific for a receptor-ligand complex of the invention.
In yet another aspect, the invention provides a method for affecting or modulating axonogenesis in a subject comprising administering to a subject an effective amount of a purified and isolated oligomerized Elk-related receptor tyrosine kinase, or an isoform or an extracellular domain thereof, a substance or compound identified using a method of the invention, or an antibody specific for a receptor-ligand complex of the invention.
The invention also relates to a pharmaceutical composition which comprises a purified and isolated oligomerized Elk-related receptor tyrosine kinase, or an isoform or an extracellular domain thereof, a substance or compound identified using a method of the invention, or an antibody specific for a receptor-ligand complex of the invention, in an amount effective to stimulate or inhibit neuronal development or regeneration and a pharmaceutically acceptable carrier, diluent or excipient.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.